Functional Analysis Of Zinc Finger Protein Genes PmZAT12 And PmBBX32 In Prunus Mume Under Low Temperature Stress

Prunus mume has numerous cultivars,and colorful flower shape.Due to the restrictions of low temperature in the north,most varieties cannot grow in the open fields in winter.Therefore,breeding more cold-resistant P.mume cultivars have very important landscape application value and broad market prospect.Zinc finger proteins are a class of transcription factors with a"finger-like"domain.They regulate the growth and development and the response to low temperature stress of plants by controlling the expression of downstream genes.Based on the gene expression profile of P.mume'Xue Mei'leaves at 4°C,we cloned five C2H2 and one BBX zinc finger protein genes,and analyzed the expression changes of these genes under low temperature stress.Six overexpression vectors were constructed and transformed into Arabidopsis thaliana.Through the low temperature stress treatment of transgenic plants,their cold resistance can be preliminarily determined.The physiological index and expression levels of cold resistant genes in PmBBX32 transgenic Arabidopsis were measured.The main research results obtained are as follows:1.We cloned five C2H2-type zinc finger protein genes?PmZAT9,PmZAT10A,PmZAT10B,PmZAT11,and PmZAT12?and one BBX-type zinc finger protein?PmBBX32?from'Xue Mei'leaves.The expression levels of these genes under low temperature induction were also examined.The results showed that these six genes had different degrees of response to low temperature induction.In the C2H2-type zinc finger protein gene,PmZAT12 and PmZAT10A are responded to low temperature induction more strongly than the other three genes?PmZAT9,PmZAT10B,PmZAT11?;the BBX-type zinc finger protein gene PmBBX32 has a weak response to low temperature induction.2.Six over-expression vectors of the target gene were constructed and transformed into Arabidopsis.We obtained T₃-generation homozygous lines of PmZAT9,PmZAT12and PmBBX32,T₂-generation single-copy lines of PmZAT10A and T₁-generation lines of PmZAT10B and PmZAT11.3.Identification of low temperature resistance of transgenic Arabidopsis.Compared with the wild type,the survival rate of Arabidopsis overexpressing the PmZAT12 gene was decreased,and the plants overexpressing the PmBBX32 gene were increased,while the plants overexpressing the PmZAT9 and PmZAT10A genes were not significantly different under cold treatment.4.Detection of physiological index and expression levels of cold resistant genes in PmBBX32 transgenic Arabidopsis.Compared with wild-type Arabidopsis,overexpressing PmBBX32 plant has lower relative electrolytic leakage and ROS accumulation,and higher antioxidant enzyme activity and proline content under cold stress.It indicated that the overexpression of PmBBX32 gene reduced the damage of plants at low temperature,and it could enhance the cold resistance of plants by increasing the activities of antioxidant enzymes and proline content.In addition,the results of RT-PCR showed that overexpression of the PmBBX32 gene can up-regulate the expression of cold stress genes and SOD-related genes,so as to enhance the cold resistance of the plants.5.Flowering phenotype analysis of Arabidopsis overexpressing the PmZAT12 and PmBBX32 genes.The statistical results of morphological indexes showed that the flowering time of Arabidopsis with overexpression of PmZAT12 and PmBBX32 was significantly later than wild type,and both of transgenic Arabidopsis had dwarfing characteristics.The study found that the number of leaves of rosette leaves in transgenic Arabidopsis increased significantly compared with wild type,so it is speculated that the phenotype of transgenic Arabidopsis may be related to slow development.